| Insert + vector | Vector only | |
| Vector DNA | 200 ng | 200 ng |
| Insert DNA (E5/E22) | 146 ng/84 ng | Not added |
| 10x T4 DNA ligase buffer | 4 μl | 4 μl |
| T4 DNA ligase (400 U/μl) | 2 μl | 2 μl |
| Nuclease-free water | Make up to 40 μl | Make up to 40 μl |
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